Pests may be controlled using either chemical pesticides or biopesticides. However, because of their broad spectrum of activity, chemical pesticides may destroy non-target organisms such as beneficial insects and parasites and predators of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.
Biopesticides make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. An example of a biopesticide is a bacterium which produces a substance toxic to the infesting pest. A biopesticide is generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.
The most widely used biopesticide is Bacillus thuringiensis. Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and pest-rich environments. During sporulation, Bacillus thuringiensis produces a parasporal crystal inclusion(s) which is toxic upon ingestion to susceptible larvaea. The inclusion(s) may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the pest (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255).
The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The six major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), Diptera-specific (cryIV) (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255), Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275). Several Bacillus thuringiensis crystal delta-endotoxins are also reportedly pesticidal to Acari, Hymenoptera, Phthiraptera, Platyhelminthes, Homoptera, Blattodea, and Protozoa.
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form. Various cry genes have been cloned, sequenced, and expressed in various hosts, e.g., E. coli (Schnepf et al., 1987, J. Bacteriol. 169:4110-4118) and Bacillus subtilis (Shivakumar et al., 1986,J. Bacteriol. 166:194-204).
Amplification of cry genes has been achieved in Bacillus subtilis. The delta-endotoxin gene of Bacillus thuringiensis subsp. kurstaki HD73 has been cloned into Bacillus subtilis using an integrative plasmid and amplified (Calogero et al., 1989, Appl. Environ. Microbiol. 55:446-453). However, no increase in crystal size was observed as compared to Bacillus thuringiensis subsp. kurstaki HD73. Furthermore, no difference in pesticidal activity was reported.
The level of expression of delta-endotoxin genes appears to be dependent on the host cell used (Skivakamar et al., 1989, Gene 79:21-31). For example, Skivakumar et al. found significant differences in the expression of the cryIIA and cryIIA delta-endotoxin genes of Bacillus thuingiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium. The cryIA gene was expressed when present on a multicopy vector in Bacillus megaterium, but not in Bacillus subtilis. The cryIIA gene was expressed in both hosts, but at a higher level in Bacillus megaterium. Sections of Bacillus megaterium cells expressing these delta-endotoxin genes were examined by electron microscopy; the presence of large bipyramidal crystals in these cells was detected. However, there is no indication that these crystals are any larger than crystals found in Bacillus thuringiensis subsp. kurstaki which normally contain these genes. Results from bioassays of the Bacillus megaterium cells expressing these delta-endotoxin genes indicate that there was no increase in pesticidal activity as compared to Bacillus thuringiensis subsp. kurstaki. Indeed, five times the concentration of Bacillus megaterium than Bacillus thuringiensis subsp. kurstaki was required to obtain the same insect killing effect.
In the prior art methods, a host cell is transformed with a recombinant DNA vector carrying a DNA sequence encoding a delta-endotoxin and DNA replication sequences. The expression of the delta-endotoxin is dependent on the replication of the recombinant DNA vector in the host. When, for the purpose of producing a desired polypeptide by recombinant DNA procedures, bacterial cells are transformed with a recombinant plasmid vector which carries inserted genetic information coding for the delta-endotoxin, it has often been observed that such plasmids become unstable even though they may, in themselves, be stably inherited in the cell. This instability may either take the form of unstable maintenance of the plasmid in the cells so that the plasmid will eventually be lost from a cell population, or so that the DNA coding for the protein in question may be deleted from the plasmid. A traditional way of solving the former problem has been to grow the transformed cells under selection pressure, that is, typically in the presence of an antibiotic to which the cells in question have been made resistant due to the presence of a gene coding for a product mediating resistance to that antibiotic on the plasmid transformed to the cells. This approach, however, is neither economically feasible in large-scale production due to the high cost of the antibiotics in question, nor is it desirable for environmental reasons. The use of antibiotics in culture media also makes it more difficult to obtain product approval from health authorities and the like.
It has previously been suggested that plasmids could be stabilized by inserting into them a DNA sequence encoding a partitioning function which ensures the even distribution of plasmids to progeny cells on cell division. An alternative method of achieving the stable inheritance of cloned DNA sequences is to provide for the integration of such DNA sequences in the genome of the host bacterium. Integration of DNA sequences present on plasmid vectors may take place by the so-called "crossing-over" procedure, e.g. as described by A. Campbell, Advances Genet. 11, 1962, pp. 101-145. According to this procedure, the plasmid vector is provided with a DNA sequence which is homologous to a region on the bacterial genome, or alternatively with two homologous sequences placed on either side of the heterologous DNA sequence to be integrated. In a subsequent recombination event, the homologous sequence and adjacent sequences on the vector are integrated into the host genome at the region of homology.
In some cases, however, it has been found that the integrated DNA sequences are deleted from the cells in the absence of selection pressure, for instance by a similar type of homologous recombination event as that responsible for the integration of the DNA. In particular, it has previously been observed that recombination between homologous DNA sequences is stimulated in the proximity of replicative DNA present on or near the DNA integrated in the host cell genome, cf. Ph. Noirot et al., J. Mol. Biol. 196, 1987, pp. 39-48; and M. Young and S. D. Ehrlich, J. Bacteriol. 171(5), May 1989, pp. 2653-2656.
An object of the present invention is therefore to provide stable integration of DNA sequences into genomic DNA, e.g. the chromosome, of bacterial, particularly Bacillus thuringiensis host cells. It is also an object of the invention to create integrants of Bacillus thuringiensis strains which produce sufficient quantities of delta-endotxins. Such integrants may be useful in broadening the host range of Bacillus thuringiensis and obtaining more effective formulations of Bacillus thuringiensis.